The recent discoveries of microRNAs (miRNAs), RNA interference, short interfering RNAs, and small modulatory RNAs have revealed potentially widespread gene regulatory mechanisms that are mediated by short RNAs in animal and plant development. miRNAs are an abundant class of~22 nt endogenous noncoding RNAs. Some have been shown to play an important role in plant and animal development by controlling gene expression at the posttranscriptional level. Recently it was demonstrated that miR-181 could promote B-cell differentiation in vitro and in vivo, providing the first evidence that miRNAs have regulatory roles in vertebrate development (Chen et al., Science, 2004, 303:83). However, examples of miRNAs functioning in vertebrate development are still rare, and the molecular mechanisms through which miRNAs exert their functions are largely unclear. This research proposal addresses these fundamental questions and aims to establish the potential widespread influences of miRNA-mediated posttranscriptional gene regulatory circuitries during hematopoiesis. Through miRNA cloning and expression analyses, we have identified a set of hematopoietic miRNAs that may play important roles in lineage differentiation. We will try to define the functions of these miRNAs in various aspects of hematopoietic lineage differentiation so that we can establish broader roles for miRNAs in hematopoiesis (aim 1). With defined in vitro and in vivo differentiation assays we will be able to dissect the potential signaling pathways that are regulated by miRNAs, determine the structural and functional relationships of miRNA genes, define the progenitor cell population(s) that miRNAs act on, and delineate the cellular processes controlled by miRNAs (aim 2). Finally, we will combine computational and experimental approaches to identify the functionally relevant target genes of hematopoietic miRNAs (aim 3). Understanding the biological functions of miRNAs in hematopoiesis will shed light on the roles of miRNAs in hematological disorders and could lead to novel therapeutic strategies to correct many hematological disorders, including leukemias. The proposed research will also provide methods, insights, and inspiration to those seeking to place miRNAs into other biological processes.